The discovery of monoclonal antibodies has evolved from hybridoma technology, with the aid of which antibodies having a particular specificity and affinity can be produced in a specific manner. Combinatorial libraries developed therefrom, including screening and selection methods, have developed into standard tools for modifying the binding properties of proteins in general.
The most widespread technique for generating and screening antibody libraries was and in some cases still is the “phage display” method, in which the particular protein of interest can be expressed as a fusion polypeptide on a bacteriophage shell protein and selected by binding to immobilised or soluble biotinylated ligands. A phage which has been constructed in this manner can be regarded as a compact genetic unit which has combined in itself both the phenotypic and the genotypic properties. Phage display has been used very successfully on antibodies, antibody fragments, enzymes, DNA-binding proteins etc. Antibodies which have desired binding properties are selected by binding to an immobilised antigen in a process called “panning”. Phages which contain non-specific antibodies are washed out and the bound phages are detached and amplified in E. coli. This set-up has been employed to generate a large number of antigen-specific antibodies. Nonetheless, phage display technology has some fundamental deficiencies and difficulties which limit its use, in particular in the production of eukaryotic proteins. Thus, for example, antibodies of very high affinity can be isolated and further processed by “panning” only with difficulty. In addition, posttranslational modifications, such as e.g. glycosylation, which can influence the specificity and affinity of the antibody, are not possible with phage display methods.
An alternative is the use of lower eukaryotic systems, such as yeast. The structural similarity between B cell-displaying antibodies and yeast cell-displaying antibodies deliver a closer analogy to in vivo “affinity maturation” than in the case of filamentous phages. Since in particular eukaryotic cells, such as yeast, are capable of producing glycosylated proteins, whereas filamentous phages cannot do this, monoclonal antibodies from eukaryotic host cells should have properties which resemble human or mammalian antibodies more so than antibodies from phages. Moreover, cloning, expression and modification of antibodies in yeast in particular has proved to be effective and simple in terms of method and practicality. U.S. Pat. No. 6,699,658 describes, for example, a yeast cell surface display method with the aid of which screening and production of combinatorial antibody libraries has become possible. The said “yeast surface display” technology is based on the transfection of yeast cells with vectors which express an immunoglobulin fused to a yeast cell wall protein, employing mutagenesis in order to generate a diversity of immunoglobulin mutants and in order then to select these cells according to the desired phenotypic properties. This technology was established in 1997 by Boder and Wittrup. They succeeded for the first time in displaying scFv fragments of a combinatorial library functionally on the surface of yeast cells and in screening them by flow cytometry, and in isolating scFv fragments having an increased affinity for the antigen. This was rendered possible by the stable coupling of geno- and phenotype, since the scFv fragment was displayed as a fusion protein having a cell wall protein intrinsic to the yeast. Presumably the most important achievement arrived at by using yeast-based display technology is the direct applicability of fluorescence-activated cell sorting (FACS), which is decisive in the efficient screening of large variant libraries. A stable genotype-phenotype coupling is achieved by fusion of a heterologous protein with proteins of the outer cell wall of S. cerevisiae. The exposure of the protein thereby achieved is the prerequisite for interaction with antigens.
However, the yeast surface display just described, as developed by Wittrup and Boder, has in particular some practical disadvantages. One disadvantage is, for example, that the various proteins expressed cannot be obtained or can be only obtained unsatisfactorily with the same yeast cell. Furthermore, by the method of Wittrup the desired immunoglobulin is bound covalently to the cell wall protein and must be isolated by additional method steps.
WO 2010/005863 describes a corresponding yeast surface display system based on yeast cells of the genus Pichia pastoris, in which the immunoglobulin is bound non-covalently to the ZZ domain of protein A, wherein the fusion protein comprising the cell wall protein agglutinin or its subunits and the ZZ domain and the immunoglobulin is only expressed and secreted simultaneously in the yeast cell, in order to be displayed on the cell surface, when corresponding various promoters for expression of said proteins are switched on or off in the correct chronological order.